The overall goal of this program is to test critically the general hypothesis that myelin antigen reactive T cells and the MS risk-associated HLA-DR2 allele contribute to the pathogenesis of multiple sclerosis (MS). To this end we developed a recombinant TCR ligand (RTL), a single chain two domain human HLA-DR2 class II molecule covalently linked to the immunodominant MOG-35-55 epitope that induced long-term tolerance and reversed established clinical signs of MOG-peptide-induced EAE in transgenic (Tg) DR2-expressing mice. The initial DR2/MOG-35-55 construct (VG312) that we produced had self-adherent surfaces and tended to form stable oligomers with an average composition of 14-subunits. The effect of oligomerization on the inhibitory function of the constructs is unknown, but potentially the oligomers might have a lower functional molarity and thus less inhibitory activity in vivo than the monomers. On the other hand, the oligomers might be able to cross-link the TCRs more efficiently than monomers, resulting in more or different signaling through the TCR that might affect inhibitory activity. We have now produced a DR2/MOG-35-55 monomer (VG342) by modifying amino acid residues in the self-contact surface that will allow a functional comparison with the 14-mer VG312 construct. Additionally, we have produced constructs that contain mouse (m)MOG versus human (h)MOG peptides with different affinities for TCRs from mice with EAE, and a new monomeric form (VG342-T) that lacks the thrombin (T) cleavage site that was engineered into the peptide-joining region of the original construct. Our goal in this application is to compare these five forms of the DR2/MOG-35-55 construct for therapeutic efficacy, induction of tolerance, and effects on mouse and human MOG-35-55 specific T cell lines, clones, and a hybridoma that we recently developed from the DR2 mice. Specifically, in this application we will address the specific hypothesis that the degree and mechanism of tolerance is governed or influenced by 1) differences in the affinity of the RTL for the TCR, 2) differences in functional avidity of RTL binding to the TCR, and 3) cleavage and release of free peptide from the RTL. Finally, we will evaluate the degree of bystander suppression induced by RTLs and follow the fate of RTL-treated T cells. These are the first studies ever to evaluate the effect on tolerance of monomeric versus oligomeric TCR blockage in vivo, and will provide the necessary foundation for clinical application of these recombinant TCR ligands (RTLs) in patients with MS.